Novel hot start nucleic acid amplification

ABSTRACT

Methods and compositions for performing nucleic acid duplication and amplification reactions are provided. A single-stranded nucleic acid binding protein is selected and provided in the reaction mixture which is assembled at a low, nonstringent temperature to include all of the necessary reagents for successful nucleic acid duplication or amplification reactions. By incorporating a single-stranded nucleic acid binding protein into the reaction mixture at low temperature, the generation of nonspecific products such as amplification products is improved despite the reaction mixture having been fully assembled at a nonstringent temperature.

This application claims the benefit of U.S. provisional patentapplication Ser. No. 60/584,362 filed Jun. 30, 2004, the contents ofwhich are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention provides a method that reduces or eliminatesnonspecific primer extension products. More specifically, the methoduses single-stranded nucleic acid binding proteins to reduce oreliminate these products. This invention is contemplated to beespecially useful as a novel Hot Start method for the polymerase chainreaction (PCR).

2. Description of Related Art

Amplification of nucleic acids is of fundamental importance in modernscience. During this process, nucleic acids are duplicated or replicatedthrough coordinated, catalytic synthesis.

In general, nucleic acid amplification occurs through a process ofhybridizing (annealing or pairing) a relatively short single-strandednucleic acid (primer or oligonucleotide), to a relatively longersingle-stranded nucleic acid counterpart (target or template) that hascomplementary nucleic acid sequence. Complementary annealing refers tothe base pairs which form and are stabilized by hydrogen bonds describedby Watson-Crick pairing rules (i.e., A-T and G-C base pairs). Apolymerase can use this hybrid (or complement) to catalytically addbases or nucleotides which are present in the reaction to the 3′ end ofthe primer. The nucleotides are added such that they are complementaryto the target or template. Since the newly synthesized strand of nucleicacid is the result of nucleotides which extend the length of the primer,this process is also known as primer extension. To be extended by apolymerase, a primer strand first must be annealed to a template strand.

Although the primer(s) used in primer extension reactions are designedto be complementary to a specific portion of the template strand, undercertain conditions the primer can and will anneal to other regions ofthe template strand with which it is only partially complementary, or inrare cases, noncomplementary. As used herein, a fully complementarypairing is referred to as and is the result of specific priming and apartially complementary (or noncomplementary) pairing is referred to asand is the result of nonspecific priming. Since the polymerase cannotdiscriminate between partial versus full complements, primer extensionproducts can and will be formed from both if both are present underextension conditions. As used herein, primer extension products fromfull complements are referred to as specific products and those frompartial (or non-) complements are referred to as nonspecific products.

The degree to which a primer will hybridize to full versus partial (ornon-) complementary sequences is governed by well-known principles ofthermodynamics. A useful parameter is known as the melting temperature(T_(m)) and is defined as the temperature at which 50% of the primer andits true complement or intended target sequence is annealed. The mostcommon method to determine the actual T_(m) is to plot temperatureversus absorbance in a UV spectrophotometer (e.g., Marnur and Doty,1962, Journal of Molecular Biology 5:109-118). This empiricaldetermination is often not practical and thus theoretical methods havebeen devised to predict melting temperatures. One such method is throughan equation known as the Wallace Rule (Suggs et al., 1981, InDevelopmental Biology using Purified Genes 23:683-693). This equationstates that T_(m) (in ° C.) is approximately equal to2×(#A+#T)+4×(#G+#C), where # is the number of A, G, C, or T basespresent in the primer. Thus, a primer 20 bases long with an equal basecontent would be predicted to have a T_(m) of 2×(5+5)+4×(5+5)=60° C.

Although other factors such as salt concentration, DNA concentration,and the presence of denaturants affect the melting temperature, the maincontribution to T_(m) is from the length and base composition of theprimer. Given a defined primer sequence, the temperature of thehybridization reaction determines the amount of specific versusnonspecific priming based on thermodynamic principles. Temperaturessignificantly below the T_(m) will permit nonspecific priming whiletemperatures significantly above the T_(m) will restrict nonspecific andspecific priming (e.g., Gillam et al., 1975, Nucleic Acids Research2(5):625-634; Wallace et al., 1979, Nucleic Acids Research6(11):3543-3557). Ideally, hybridization is carried out at or near theT_(m) of the primer(s) to generate specific complements and thusspecific primer extension products. As used herein, hybridization andprimer extension temperatures significantly lower than the T_(m) of theprimers are referred to as permissive or nonstringent while temperaturesat or near the T_(m) are referred to as restrictive or stringent. Thus,permissive or nonstringent temperatures lead to nonspecific primerextension products while restrictive or stringent temperatures lead tospecific ones.

A well-known example of primer extension is the polymerase chainreaction (PCR). In this technique, DNA synthesis occurs in a series ofsteps comprising a cycle, this cycle being repeated many times toamplify the primer extension reaction products for further analyses. Twoprimers typically are used in which their respective 3′-ends face oneanother to generate a double-stranded DNA product whose length isdefined as the distance between the primers. Typically, the cycleconsists of a step which generates single-stranded DNA, a step whichallows primers to hybridize with their target sequences, and asubsequent step for primer extension by the polymerase. The PCRtechnique is described in detail in U.S. Pat. Nos. 4,683,202; 4,683,195;and 4,965,188. A variant of PCR, which is called reversetranscription-PCR (RT-PCR), is when RNA is used as a template in thereaction instead of DNA. In this technique, an initial step ofconverting the RNA template to DNA is performed with a polymerase whichhas reverse transcriptase activity. Following this initial templateconversion (reverse transcription step), reactions proceed as instandard PCR.

Each cycle of PCR generates a geometric expansion of the original target(i.e., doubling per cycle), which after the 25-50 cycles typicallyemployed in PCR can amplify the target well over a billion times.Unfortunately, amplification from nonspecific priming can also occurwhich is detrimental since these nonspecific products may obscurespecific ones. The specificity of the PCR depends on many factors, butas previously discussed, the temperature of the hybridization andsubsequent extension steps is important in obtaining specific primerextension products. Fortunately, the discovery and widespread use ofthermostable polymerases, such as the polymerase from Thermus aquaticus(Taq DNA Polymerase), allows the use of more stringent reactiontemperatures (Chien et al., 1976, Journal of Bacteriology127(3):1550-1557; Saiki et al., 1988, Science 239(4839):487-491).Stringent hybridization temperatures increase the probability ofgenerating specific products.

Although the temperatures used during the polymerase chain reaction canbe stringent, the reaction mixtures themselves are not convenientlyassembled at higher temperatures, temperatures at which greater primingspecificity occurs. PCR reactions are usually assembled at lowertemperatures such as on ice or most preferably at room temperature(i.e., 20-25° C.). If the average primer can be assumed to have a T_(m)of about 50-60° C., the temperatures at which reaction set-up occur areclearly significantly lower and will favor nonspecific priming. At roomtemperature, the conventional polymerases used in the PCR (e.g., Taq DNAPolymerase) have some degree of catalytic activity which leads to thesynthesis of nonspecific reaction products. In addition, even if thereactions are assembled on ice, they must be placed in a machine whichprovides the temperatures necessary for cycling. Stringent hybridizationtemperatures higher than ice cannot be achieved instantaneously andnonspecific products can also be generated during this “ramping” stage.At permissive temperatures primers not only pair nonspecifically withthe template but also pair with other primers leading to nonspecificprimer extension products known as “primer-dimers.” Nonspecificamplification is a ubiquitous problem during the assembly of polymerasechain reactions and is covered in greater detail in Chou et al., 1992,Nucleic Acids Research 20(7):1717-1723.

Since nonspecific amplification products can be generated duringassembly of PCR reactions, a method is needed that can reduce oreliminate these artifacts. Various methods have been developed toaddress this problem. These techniques are generally known as“hot-start” methods because the primer extension reactions are notallowed to “start” until stringent or “hot” hybridization temperatureshave been reached. Several of these methods are briefly described below.

In the simplest hot-start method, one of the critical components forsuccessful DNA synthesis is omitted from the reaction mixture duringpreparation at room temperature. Then, the omitted component is addedmanually, as through pipetting, after the temperature of the reactionmixture has reached, or more usually exceeded, a threshold stringenttemperature based upon the T_(m) of the primer(s). This method is oftencalled manual hot-start PCR. For example, one may omit the polymerase orthe divalent cation (e.g., Mg²⁺) which is essential for polymeraseactivity from the reaction mixture until the stringent temperature isreached or exceeded. Because a key component is unavailable at lowertemperatures, nonspecific extension products cannot be formed. Thismethod is tedious and cumbersome when multiple reactions are performedand also can lead to contamination of PCR reactions since tubes in closeproximity to one another must be opened and closed manually by theoperator in order to introduce the omitted component.

In another hot-start method, all of the necessary components areassembled in the reaction mixture at room temperature, but one criticalcomponent is physically isolated from the remainder of the reactionmixture using a barrier material that will melt or dissolve at elevatedtemperatures. Once the barrier material, typically a wax, has melted,the isolated component is introduced into the remainder of the reactionmixture and the primer extension reaction can proceed at the morestringent temperature. Conventionally, the polymerase is isolated usingthe barrier or wax material. This method, which is described in detailin U.S. Pat. No. 5,411,876 and Chou et al., Nucleic Acids Research20(7):1717-1723 (1992), allows more specific amplification but iscumbersome in the set-up and implementation of the barrier material.

Another method is to use an antibody that non-covalently binds to thepolymerase and prevents its activity at lower temperatures. At highertemperatures, the non-covalent bond between the antibody and thepolymerase is disrupted and polymerase activity is restored for the restof the PCR reaction. This method is further described in U.S. Pat. No.5,338,671. Although this method is effective, the production process forgenerating the antibody is expensive and can introduce contaminatingmammalian genomic DNA into the PCR reaction.

Yet another technique involves covalent attachment of a chemical moietyto the polymerase which blocks its activity at lower temperatures. Thiscovalent bond can be broken after significant heating (e.g., above 95°C. for about 10-15 minutes) after which the polymerase activity isrestored. A variety of chemical modifications can be introduced toproduce the polymerase-moiety complex required to practice thistechnique as described in U.S. Pat. Nos. 5,677,152, 6,183,998 and6,479,264. This technique has the disadvantage of requiring an extensiveinitial heating step which can damage DNA through heat-induceddepurination. Such an extensive heating step also markedly reduces theactivity of the polymerase relative to standard PCR methods.

In summary, primer extension reactions can be defined by two key events.One, the process of hybridizing the primer to the template and two, theextension of the hybrid by the catalytic action of a polymerase. Thespecificity of the hybridization is governed by the principles ofthermodynamics in which lower temperatures favor nonspecific priming andamplification artifacts. Because polymerase chain reactions areconventionally assembled at lower temperatures, amplification artifactscan be a problem. Various methods have been developed to address thisproblem, techniques known as hot-start PCR. The present invention is anovel method of hot-start PCR.

SUMMARY OF THE INVENTION

A method of duplicating a template nucleic acid, or a portion thereof,is provided wherein a primer having a nucleotide sequence that iscomplementary to a target portion of the template nucleic acid ishybridized to the template nucleic acid and then extended via an enzyme.The method includes the following steps: (a) at a first temperature,preparing a reaction mixture including a primer, a template nucleicacid, an enzyme effective to catalyze primer extension and an effectiveamount of single-stranded nucleic acid binding protein, (b) at a secondtemperature higher than the first temperature, carrying out ahybridization reaction to produce a hybridized product, and (c) at athird temperature higher than the first temperature, carrying out aprimer extension reaction to produce from the hybridized product anextended product; wherein the generation of specific extended product isimproved as a result of incorporating the single-stranded nucleic acidbinding protein into the reaction mixture at the first temperature.

A primer complex also is provided. The complex includes a primer havinga nucleotide sequence that is complementary to a specific target portionof a template nucleic acid molecule, and a single-stranded nucleic acidbinding protein interacting with the primer. The single-stranded nucleicacid binding protein is selected such that 1) it in effect inhibits theprimer from participating in a primer extension reaction up to at leasta first temperature at or below 30° C., and 2) that interaction ceasesor is disrupted at a second temperature in the range of 30° C. to about72° C. such that the primer is substantially uninhibited by thesingle-stranded nucleic acid binding protein from participating in aprimer extension reaction at the second temperature.

A PCR reaction mixture also is provided, including a primer having anucleotide sequence that is complementary to a specific target portionof a template nucleic acid, and a single-stranded nucleic acid bindingprotein effective to inhibit the primer from participating in a primerextension reaction up to at least a first temperature at or below 30°C., wherein the inhibitive capability of the single-stranded nucleicacid binding protein is lost at a second temperature in the range of 30°C. to about 72° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an agarose gel electrophoresis image illustrating theeffectiveness of hot-start methods using single-stranded DNA bindingproteins according to the disclosed methods, compared to other methodsas described in Example 1.

FIG. 2 is an agarose gel electrophoresis image illustrating theeffectiveness of hot-start methods using a mixture of wild-type and theΔ26C mutant of T7 SSB, T7 gp2.5-Δ26C, as described in Example 2.

FIG. 3 a is a schematic of the polymerase blocking assay of Example 3.The forward primer has a HEX label attached to its 5′end to allowfluorescent detection. The primer extension product is a 27 baseaddition to the 23-base forward primer. The observed change duringdenaturing PAGE is from 23 to 50 bases.

FIG. 3 b is a denaturing, polyacrylamide gel electrophoresis imageillustrating the blocking effects of a mixture of wild-type and the Δ26Cmutant of T7 SSB in the mock PCR reaction described in Example 3.

FIG. 4 is an agarose gel electrophoresis image illustrating a range ofconcentrations of wild-type T7 SSB that are effective in a hot-startmethod as herein described, Example 4.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

As used herein, when a range such as 5-25 (or 5 to 25) is given, thismeans preferably at least 5 and, separately and independently,preferably not more than 25.

Also as used herein, a ‘single-stranded nucleic acid binding protein’(SSB or SSBs when plural) is a polypeptide or protein that exhibits veryhigh affinity for interacting with (i.e., binding) single-strandednucleic acids. Typically, a single-stranded nucleic acid binding proteinexhibits a higher affinity for and preferentially binds tosingle-stranded nucleic acids over double-stranded nucleic acids. SSBscan bind to a single-stranded molecule or fragment of DNA or RNA, butgenerally a specific type of SSB prefers one to the other. The SSBproteins discussed herein have a higher affinity for DNA than for RNA,and are more often referred to as single-stranded DNA binding proteinsin the scientific literature. SSBs bind to single-stranded nucleic acidsstoichiometrically, which means that they bind in approximately fixedmolar ratios with respect to the nucleic acid. In addition, SSBsgenerally bind nucleic acid with no sequence specificity (i.e., withoutregard to the base composition of the nucleic acid). The SSBs referredto herein are not enzymes, meaning they do not exhibit any substantial(or known) enzymatic activity (Chase and Williams, 1986, Annual Reviewsof Biochemistry 55:103-136).

Also as used herein, the term ‘hybridization’ refers to the bonding ofone single-stranded nucleic acid to another single-stranded nucleicacid, such as a primer strand to a template strand, via hydrogen bondsbetween complementary Watson-Crick bases in the respectivesingle-strands to thereby generate a double-stranded nucleic acid hybridor complex as otherwise known in the art. Commonly, the terms‘hybridize,’ ‘anneal,’ and ‘pair’ are used interchangeably in the art todescribe this reaction, and so too they are used interchangeably herein.Hybridization may proceed between two single-stranded DNA molecules, twosingle-stranded RNA molecules, or between single-strands of DNA and RNA,to form a double-stranded nucleic acid complex.

Also as used herein, the term ‘denaturation’ means the process ofseparating double-stranded nucleic acids to generate single-strandednucleic acids. This process is also referred to as ‘melting’. Thedenaturation of double-stranded nucleic acids can be achieved by variousmethods, but herein it principally is carried out by heating.

Also as used herein, the term ‘single-stranded DNA’ will often beabbreviated as ‘ssDNA’, the term ‘double-stranded DNA’ will often beabbreviated as ‘dsDNA’, and the term ‘double-stranded RNA’ will often beabbreviated as ‘dsRNA’. It is implicit herein that the term ‘RNA’ refersto the general state of RNA which is single-stranded unless otherwiseindicated.

Also as used herein, an SSB is said to ‘interact’ or to be ‘interacting’with a primer when it cooperates with, or otherwise is correlated,associated, coupled or otherwise complexed to, the primer in such amanner so as to substantially inhibit or prevent the primer fromparticipating in a primer extension reaction. The term ‘interact’ andvariants thereof is/are considered to include, but not necessarily to belimited to, a chemical bond (covalent, non-covalent or otherwise) aswell as other modes of binding or bonding that are or may be achievedbetween an SSB and its associated primer so as to produce the primerextension-inhibitive effect described in this paragraph, and furtherdescribed herein as well as observed in the following Examples.

The present invention provides methods and reagents that inhibit orprevent the generation of nonspecific primer extension products thatresult from nonspecific priming events at permissive temperatures. Themethods are envisioned to be particularly useful and applicable toprimer extension via the polymerase chain reaction (PCR) although theinvention is not limited to such reactions. The present invention isapplicable to any reaction or process incorporating otherwiseconventional hybridization and primer extension reactions to produce anamplified or newly synthesized double-stranded product from aprimer-template hybrid, whether as an intermediate or final product. Assuch, the present invention would also be useful for the variation ofPCR called reverse transcription-PCR in which a reverse transcriptionstep converts RNA to DNA. Other examples of primer extension reactionsinclude DNA and RNA sequencing, reverse transcription, in vitrotranscription and isothermal amplification, among others.

PCR mixtures usually are prepared or assembled at room temperature (lessthan 30° C., more typically 20-25° C.) or on ice (0° C.) in reactiontubes suitable for accommodating the hybridization and primer extensionreactions in a conventional thermal cycler. Nearly, if not entirely, allPCR mixtures initially are prepared below 37° C. A typical PCR mixturewill include at least the following essential components:

-   -   a template nucleic acid, which can be single-stranded or        double-stranded, which it is desired to amplify;    -   at least one primer that is complementary to a target portion of        the template nucleic acid—if the template is double-stranded and        it is desired to amplify both strands then at least two primers        will be provided, each being complementary to a specific target        portion on each of the sense and anti-sense template strands;    -   the four deoxyribonucleotides necessary for enzyme-directed        nucleic acid synthesis (dATP, dGTP, dTTP and dCTP), occasionally        exogenous nucleotides may be included as well (e.g., dUTP);    -   an enzyme or enzymes for directing nucleic acid synthesis,        typically a polymerase such as Taq DNA polymerase and/or other        thermostable polymerases, reverse transcriptase (e.g., MMLV-RT        or AMV-RT) or other suitable enzyme if template is RNA;    -   where a polymerase(s) is used, a divalent cation such as Mg²⁺,        Mn²⁺, etc., which is an accessory for polymerase activity;    -   a suitable reaction buffer solution capable of supporting the        cyclic hybridization and primer extension reactions as further        described below.

All of the foregoing components are conventional for PCR (and RT-PCR)and the amounts of each (as well as compositions for the reaction buffersolution) are well known or ascertainable to those having ordinary skillin the art without undue experimentation. Accordingly, they will not befurther described here except to note that in conventional hot-start PCRtechniques such as those described in the BACKGROUND section, at leastone of the foregoing essential components, typically the polymerase(s),is withheld or isolated from the remaining mixture until a stringenthybridization temperature has been reached. In the present invention,all of the foregoing essential components can be assembled together inthe reaction mixture at room temperature, along with an effective amountof a SSB(s) as hereinafter described, yet nonspecific primer extensionproducts are inhibited or prevented from occurring at nonstringenttemperatures such as room temperature. Of course, other components whichare known or conventional in the art also can be included in thereaction mixture to achieve various known or conventional effects, e.g.,glycerol, betaine, DMSO, detergents, etc., the selection andincorporation of which are within the ability of one having ordinaryskill in the art.

Once the PCR mixture has been prepared (all reaction componentsintroduced into the reaction tubes in the appropriate concentrations),the tubes can be transferred to a thermal cycler to carry out the cyclicreactions for automated PCR. Less preferably, manual PCR can be used. Apreferred PCR temperature profile contemplated herein is disclosed inTable 1. TABLE 1 Steps for the Polymerase Chain Reaction Step No. StepName Temperature Time Effect 1 Initial 92-95° C. 0.5-5 Denaturedouble-stranded DNA Denaturation minutes template. 2 Denaturation 92-95°C. 1-60 Denature dsDNA. seconds 3 Hybridization 50-72° C. 1-60 Primersbind to complementary target seconds portions of template nucleic acidstrands. 4 Extension 68-72° C. can vary, Polymerase extends primerthereby generally synthesizing new strand complementary about 0.5-20 totemplate strand to form dsDNA. minutes Repeat steps 2-4 as necessary,generally 25-45 times to amplify template nucleic acid. 5 Final 68-72°C. 5-10 Ensure full-length primer extension Extension minutes products.6 Final Soak 4-10° C. as Storing of reaction products until necessaryneeded.

The steps (and resulting products) described in Table 1 will be familiarto those having ordinary skill in the art, so they are only brieflydescribed here. As will be understood, the Initial Denaturation step iscarried out to heat-denature double-stranded template strands, and isnot repeated as a part of the cycle. The cycle which is repeated manytimes consists of the following steps. During the Denaturation step,which is conventionally shorter in duration than the InitialDenaturation step, dsDNA is heat-denatured to generate ssDNA which canbe annealed in the subsequent step. During the Hybridization step, theprimer and template strands are annealed at a stringent temperature soas to produce, preferentially, a specific hybridization product, ascompared to a nonspecific hybridization product which would result ifhybridization were carried out at a lower, nonstringent temperature.Next, during the Extension step, a primer extension reaction is carriedout at a temperature that preferably has been optimized for theparticular enzyme or enzymes being used to catalyze the primer extensionreaction. The foregoing cycle of steps is repeated many times (e.g.25-45 times) to generate an amplified double-stranded primer extensionproduct.

In the variation of PCR known as reverse-transcription PCR (RT-PCR), anadditional step is carried out before the Initial Denaturation step toconvert the RNA substrate to DNA by the action of a RNA-dependent, DNApolymerase (reverse transcriptase). This enzymatic conversion can beaccomplished by a thermostable polymerase other than Taq DNA Polymerase(e.g., Tth DNA Polymerase) or more commonly by less thermostablepolymerases such as MMLV-RT or AMV-RT. This step typically requirestemperatures from 37-75° C. and times from 1-60 minutes. Following thisinitial template conversion step, the reactions proceed as outlinedabove.

The times and temperatures disclosed for the steps in Table 1 are notmandatory and are intended merely as a useful guide to selectappropriate conditions. Selection of appropriate cycle step times andtemperatures is well within the ability of a person having ordinaryskill in the art depending on the particular nucleic acid to beamplified, the polymerase to be used, as well as other reaction-specificfactors. Several of the steps in Table 1 may be omitted depending onfactors well recognized by those having ordinary skill in the art.Others can be optimized for time or temperature depending onreaction-specific factors such as those mentioned above.

For example, the Final Extension step often is omitted. Also, theoptimal temperature for Taq DNA Polymerase (and other thermostablepolymerases) during the Extension step generally is between about 68-74°C. It is noted further that the Hybridization and Extension steps inTable 1 can be performed at the same temperature, simultaneously;alternatively the Extension step can be performed at a highertemperature than the Hybridization step. It is seen in Table 1 that theExtension step preferably is carried out within the temperature range of68-72° C. However, this step can be carried out substantially in thesame range of temperature as the Hybridization step; that is from 50° C.to about 72° C. depending on reaction-specific factors, particularly thepolymerase or other enzyme that is used to facilitate the synthesis(primer extension) reaction during the Extension step. Alternatively,the Hybridization and Extension steps can be carried out at atemperature lower than 50° C. so long as such lower temperature issufficiently stringent to produce hybridization, and consequentextended, products of desired specificity.

The following further points are noted for completeness:

-   -   1) Denaturation temperatures typically are less than 100° C. but        greater than 90° C.; incubation time may be 1 second up to about        15 minutes. These temperatures and times are chosen to        sufficiently denature dsDNA to produce ssDNA.    -   2) Hybridization temperatures typically are about or less than        72° C. but greater than 50° C., with the specific temperature        selected depending on the melting temperature (T_(m)) of the        primer(s) to provide high stringency.

Herein, a single-stranded nucleic acid binding protein (SSB) isincorporated into a primer extension reaction mixture at a lowtemperature (such as room temperature) that is nonstringent as to thegeneration of nonspecific primer extension products. The effect is thatdespite the presence in the reaction mixture, at low temperature, of allthe necessary components for successful hybridization and primerextension reactions, the formation of specific primer extension productsnonetheless is improved compared to nonspecific products. This effect isbelieved to result from the SSB binding to single-stranded nucleic acidsin the reaction mixture at low, nonstringent temperatures that are morepermissive for nonspecific primer extension products. Specifically, theSSB in effect sequesters the primers (which are single-stranded nucleicacids) in the reaction mixture at low, nonstringent temperatures atwhich these reaction mixtures typically are prepared.

It is believed the incorporation of SSB(s) into the primer extensionreaction mixture at nonstringent temperatures may prevent or inhibit twodifferent events. First, SSBs may prevent or inhibit primers fromhybridizing to other single-stranded nucleic acids due to their bindingto the primers to form an SSB-primer complex at low, nonstringenttemperatures. Second, if a primer-template hybrid were to be formed,SSBs may prevent primer extension by blocking access of the polymeraseto the primer strand's 3′-end, e.g., if SSB remains bound at least tothe hybridized primer's 3′-end. This would inhibit the polymerase'sability to assemble nucleotides to that strand for carrying out theextension reaction.

It is noted the invention is not to be limited to either of theforegoing mechanisms, which are believed, but not certain, to beresponsible in whole or in part for the observed behavior. Indeed, theremay be alternative explanations as to the mechanism for the reducedgeneration of nonspecific primer extension products. What is evident isthat the SSB(s) interacts with the primer(s) at nonstringenttemperatures in some manner (e.g., through binding) so that the primersthereby are prevented, or at least inhibited, from participating inprimer extension reactions at those temperatures. It further has beenshown that such inhibitive interaction between SSB and the primers canbe reversed through heating to an elevated temperature that is morestringent for primer-template hybridization as described more fullyherein.

The present methods are referred to by the inventors as ‘primersequestration’ because the primers are believed to be (or at least theeffect is as though they are) sequestered, and thus prevented orinhibited from participating in primer extension reactions atnonstringent temperatures. Following preparation of the primer extensionreaction mixture including all the necessary components including theSSB at low temperature, the temperature of the mixture is elevated inaccordance with the amplification reaction cycle profile, e.g., asdescribed in Table 1, to perform a desired amplification reaction. TheSSB is selected such that at the temperature at which the reactions areto proceed (Hybridization and Extension steps in Table 1), the SSB is orbecomes denatured, or otherwise ceases to interact with or becomesdissociated from the primers, as by breaking or disrupting a chemical orphysical bond therebetween, thereby releasing the primers so they arefree to participate in the reaction. Moreover, such temperatures (50-72°C. from Table 1) are stringent compared to the temperature at which thereactions were assembled, so specific annealing is thermodynamicallyfavored over nonspecific annealing.

In one embodiment, SSBs are selected which are effective to interact orassociate with the primers via a thermolabile (i.e., heat-sensitive)interaction. This interaction is spontaneously disrupted at elevatedtemperatures, preferably at or near the range of more stringent yetoptimal temperatures for polymerase activity (typically 50-75° C., morepreferably 68-72° C.), but preferably not less than 30, preferably 37,preferably 40, preferably 50, degrees Celsius. In a preferredembodiment, the bond between the SSB and the single-stranded nucleicacid is a non-covalent bond that is sensitive to heating (i.e., aboveabout 30° C., 40° C. or 50° C.). When the temperature of the reactionmixture is elevated above these temperatures, temperatures which favorspecific priming, the thermolabile interaction is terminated and theprimers may participate in the hybridization and subsequent extensionreactions.

Alternatively, the SSBs may bind to and thereby sequester the primermolecules at a temperature at or below 30° C., but become denatured atan elevated temperature in the range of 30° C. to 98° C. or 50° C. to98° C. (more preferably up to 96° C., more preferably up to 95° C.) suchthat the interaction between the primer and the SSB is terminated orcaused to be terminated as a result of or in conjunction with thedenaturation of the SSB, thereby releasing the associated primers suchthat they are free to anneal to their intended targets. In this manner,the primers are sequestered at lower, nonstringent temperatures wherehybridization specificity is relatively low, but are free to formhybrids at elevated temperatures where stringency and consequentlyhybridization specificity are relatively high.

It is envisioned that any SSB or combination of SSBs may be useful inthe present invention with the preferred (but not limited to)characteristics: 1) the SSB(s) binds primers at lower temperaturescommonly used during assembly of PCR reactions (i.e., at, near or lowerthan room temperature, or between 0-30° C., more typically between15-27° C.); 2) the SSB(s) binds primers in commonly used or conventionalPCR buffers; and 3) the SSB(s) does not bind primers at more stringenttemperatures for specific hybridization (preferably at about or greaterthan 30, 40, 50, 60, 70, 80, or 90, degrees Celsius). Termination of theinteraction between the SSB(s) and the primers at elevated temperaturesmay be due to a thermolabile bond or otherwise via denaturation of theSSB(s). This makes the primers available during the operative steps of aPCR and viable to be extended by the polymerase or polymerases in thereaction mixture.

In a preferred embodiment, the SSB used in the disclosed methods iswild-type T7 SSB, a mutant variant of T7 SSB, or a combination thereof.Wild-type T7 SSB is also known as T7 gp2.5 or T7 gene 2.5 in thescientific literature, a term that describes its coding sequence'sposition in the bacteriophage T7 genome. The term ‘wild-type’ hereinmeans the non-mutated or original DNA and protein sequence provided inpublicly available databases and literature (e.g., Dunn and Studier,1983, Journal of Molecular Biology 166(4):477-535). T7 SSB forms stabledimers in solution which are composed of two identical subunits thathave a molecular weight 25,562 gm mol⁻¹ each. T7 SSB binds with highaffinity to ssDNA over dsDNA and each protein monomer binds a length ofabout 7 nucleotides. The thermostability of T7 SSB has been determinedand its melting temperature (T_(m)) is about 53° C. The meltingtemperature of a protein is analogous to the melting temperature ofdsDNA and is defined as the transition temperature at which about 50% ofthe protein is completely denatured relative to its native state.Herein, an SSB is termed ‘denatured’ when it is or ceases to beeffective to prevent or inhibit the generation of primer extensionproducts according to the disclosed methods, for example because it haslost its ability to bind to single-stranded nucleic acids as by heatingto unwind the protein from its native or effective conformation. Athorough characterization of T7 SSB is found in Kim et al., 1992,Journal of Biological Chemistry 267(21):15022-15031.

SSBs are known to bind to single-stranded nucleic acidsstoichiometrically. In order to produce the inhibitive effect atnonstringent temperatures as described herein, it is preferred the SSBconcentration provided in a reaction mixture be sufficient to produce astoichiometric excess of SSB relative to the primers in the mixture.Determination of the stoichiometric ratio between a particular SSB and aparticular primer (or primers) is well within the ability of one havingordinary skill in the art without undue experimentation, and in fact thestoichiometric ratios for numerous SSBs are known from the publishedliterature. In addition, most single-stranded DNA binding proteins,including the wild-type and mutant T7 SSBs discussed herein, have abinding affinity for ssDNA that is generally a few orders of magnitudegreater than their affinity for dsDNA or RNA (e.g., Chase and Williams,1986, Annual Reviews of Biochemistry 55:103-136; Lindberg et al., 1989,Journal of Biological Chemistry 264(21):12700-12708; Curth et al., 1996,Nucleic Acids Research 24(14):2706-2711). Thus, calculations andExamples that follow, dsDNA and/or RNA template amounts in the reactionare not taken into consideration. This approximation applies to moststandard PCR reactions since generally dsDNA is the preferred or mostcommon template.

As an example, T7 SSB (wild-type and mutant varieties) interacts withabout 7 single-stranded nucleotide bases of DNA per protein molecule(also referred to as monomer). For a primer having a length of 21nucleotide bases, this equates to a stoichiometric ratio of 3 monomersof T7 SSB per molecule of primer. Depending on the concentration of theprimer and the molecular weight of the protein, an appropriateconcentration for the SSB can be determined through simple arithmetic toproduce a desired stoichiometric excess of SSBs. As evidenced in Example4 below, it is desirable to have at least a 50 percent stoichiometricexcess of SSBs versus primers in the reaction mixture, or astoichiometric ratio of 1.5. Even more preferred is a 100 percentstoichiometric excess (a 1-fold excess, stoichiometric ratio of 2).Still more preferred is a 2-fold, 3-fold, or 4-fold excess of SSBsversus primers, corresponding to stoichiometric ratios of 3, 4 and 5,respectively.

For the primer sequestration methods disclosed herein, the wild-type ornaturally occurring T7 SSB is preferred. For convenience, the amino acidsequence of wild-type T7 SSB is provided in the Sequence Listing as SEQID NO. 4; the DNA gene sequence that codes for wild-type T7 SSB also isprovided as SEQ ID NO. 3. In addition to the wild-type protein, themutants T7 gp2.5 Δ21C (SEQ ID NO. 5), T7 gp2.5 F232L (SEQ ID NO. 7) anda mixture of wild-type and T7 gp2.5 Δ26C (SEQ ID NO. 6) also have provenuseful as will be shown in the following Examples, and also arepreferred. T7 SSB mutants Δ21C (SEQ ID NO. 5) and Δ26C (SEQ ID NO. 6)have a deletion of the last 21 and 26 amino acids of the wild-typeprotein, respectively. They have been shown to bind single-stranded DNAwith at least 10-fold greater affinity over the wild-type protein (e.g.,T. Hollis et al., 2001, Proceedings of the National Academy of Sciences98(17):9557-9562; Rezende et al., 2002, Journal of Biological Chemistry277(52):50643-50653; Hyland et al., 2003, Journal of BiologicalChemistry 278(9):7247-7256; He et al., 2003, Journal of BiologicalChemistry 278(32):29538-29545). T7 SSB mutant F232L (SEQ ID NO. 7) is achange of the 232^(nd) amino acid of the protein from phenylalanine toleucine and has been previously shown to bind single-stranded DNA withabout 3-fold greater affinity than the wild-type protein (He et al.,2003, Journal of Biological Chemistry 278(32):29538-29545). It is notedthat other mutants of T7 SSB not listed herein also may be useful in thedisclosed methods.

In less preferred embodiments, certain mutant E. coli SSBs and T4 SSB(both wild-type and mutant varieties) also may be useful to provide asufficient primer sequestration effect at nonstringent temperatures asdescribed herein, e.g., through reversible interaction (such as binding)with the primers at those temperatures. It is noted that wild-type E.coli SSB has been found to be unsuitable for use in the disclosedmethods because it has been shown to interfere with PCR (see Example 1).When wild-type E. coli SSB is used in stoichiometric excess over theprimers, PCR amplification products are not observed. Thus, this SSBappears to continue to bind or interact with the primers even at theelevated, more stringent temperatures required for specificprimer-template hybridization. A potential explanation is that it iswell-known E. coli SSB retains some binding activity even after boilingfor up two minutes (Chase and Williams, 1986, Annual Reviews ofBiochemistry 55:103-136). Thus, unlike T7 SSB, wild-type E. coli SSBappears to be able to withstand exposure to high temperatures and itsinhibitive effects on primer extension reactions are not readilythermally inactivated.

Other SSBs not particularly described herein are suitable for use in thepresent invention so long as they meet the criteria outlined previously.

The inventors have cloned the nucleotide sequence for, and expressed andpurified protein from, wild-type and mutant forms of T7 SSB as describedbelow. The following procedures are well within reasonable standards forthose of ordinary skill in the art. The growth and purificationprocedures can be modified from those described below depending on thebinding protein being purified as well as contaminants present in thepreparation.

Preparation of Wild-Type and Mutant Variety T7 SSBs

Construction of T7 SSB expression plasmid—Two primers, a 5′-end primerwith a Nde restriction site (5′-ATC-{overscore(CAT-ATG)}-GCT-AAG-AAG-ATT-TTC-ACC-TCT-GCG-3′, SEQ ID NO. 1) and a 3′end primer with Sal1 and Xma1 restriction sites (5′-{overscore(GTC-GAC)}-{overscore(CCC-GGG)}-TTA-GAA-GTC-GCC-GTC-TTC-GTC-TGC-TTC-C-3′, SEQ ID NO. 2) wereused to PCR-amplify the wild-type T7 SSB gene nucleotide sequence frompositions 9158-9856 in purified bacteriophage T7 genomic DNA (USBCorporation, Cleveland, Ohio). The complete genome sequence for T7 canbe found at locus NC_(—)001604 at the National Center for BiotechnologyInformation (NCBI). Bacteriophage T7 is publicly available from theAmerican Type Culture Collection (ATCC) under catalog numbers 11303-B38™and BAA-1025-B2™. The sequences for wild-type T7 gene 2.5, itscorresponding protein (wt T7 gp2.5, also referred to as T7 SSB) and themutant varieties thereof which are discussed herein are provided in theSequence Listing for convenience and ease of reference.

PCR generated DNA fragments (wild-type T7 SSB gene) were ligated intoTOPOII™ vector (Invitrogen Corporation), transformed into TOP10™chemically competent E. coli (Invitrogen Corporation) and the resultingplasmid containing the wild-type T7 SSB gene (SEQ ID NO. 3) was selectedin presence of kanamycin. The clone generated from PCR-amplified DNA wassequenced and found to be free of mutations. The plasmid was then cutwith Nde1 and Xma1 and cloned into the pRE expression vector. Thisexpression vector is under the control of the powerful promoter pL fromthe bacteriophage λ which is repressed by the λ repressor at 30° C. Theexpression from the pL containing vector is induced by raising thetemperature to 42° C. The resulting plasmid containing T7 SSB (SEQ IDNO. 4) was selected in presence of ampicillin. All mutant forms of T7SSB prepared herein were expressed from the base DNA clones described inthis paragraph, which were first altered using reverse primers thateither incorporated base changes to alter amino acids or introduced astop codon to terminate protein synthesis at the desired locationdepending on the mutation to be prepared.

Growth and purification of T7 SSB—The plasmid pRE containing thewild-type or mutant varieties of T7 SSB prepared herein, under thecontrol of λ promoter, was grown overnight at 30° C. in 500 ml TerrificBroth and 100 μg/ml ampicillin. This culture was used to inoculate 10liters of TB and 50 μg/ml ampicillin in a New Brunswick fermentor. Thecells were incubated with aeration at 30° C. At a cell densitycorresponding to A₅₉₀=1.53, the cells were induced by raising thetemperature to 42° C. to induce the expression of T7 SSB. Afterinduction, the cells were incubated for 2 additional hours and thenharvested by centrifugation at 6,000 rpm for 15 minutes in a SorvallGS-3 rotor. The cell paste (83 gin) was then stored at −80° C.

Preparation of cell extract—20 gm of frozen cells were thawed in 80 mlof 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% sucrose, 100 mM NaCl, 2 mMPMSF and 10 ml of lysozyme (10 mg/ml) were added. After incubation ofthe mixture for 30 minutes on ice with constant stirring, 21 ml of 5MNaCl were added to bring the final concentration of NaCl to 1 M. Thecells were then heated in a 37° C. water bath with constant stirringuntil the temperature reached 20° C. and then cooled in an ice waterbath until the temperature was reduced to 4° C. The lysate was thencentrifuged for 45 minutes at 40,000 rpm in a Beckman Ti-45 rotor. Thesupernatant (122 ml) was Fraction I.

DEAE Cellulose chromatography—A column of Whatman DE52 DEAE cellulose(19.6 cm²×5 cm) was prepared and equilibrated with 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 10% glycerol (Buffer A) containing 350 mM NaCl.Fraction I was diluted with Buffer A to give a conductivity equivalentto Buffer A containing 350 mM NaCl. The diluted Fraction I (˜350 ml) wasapplied to the column. T7 SSB is not retained under these conditions.The flow through and wash fractions (˜400 ml) were pooled to givefraction II.

Ammonium Sulfate Precipitation—To 400 ml of fraction II, ammoniumsulfate was added to 75% saturation (203 gm) over a period of 60 minutesand was stirred slowly for an additional 60 minutes. The precipitate wascollected by centrifugation at 14,000 rpm for 45 minutes in a SorvallGSA rotor and dissolved in 50 ml of Buffer A containing 25 mM NaCl anddialyzed overnight against the same buffer (Fraction III).

Heparin Sepharose CL-6B Chromatography—A column of Heparin (0.64 cm²×12cm) was prepared and equilibrated with Buffer A containing 25 MM NaCl.Fraction III was applied to the column and eluted with a linear gradientfrom 25 mM to 1M NaCl. The fractions were analyzed on SDS-PAGE and thefractions (134 ml) containing the T7 SSB were pooled and dialyzedovernight against Buffer A containing 100 mM NaCl (Fraction IV).

DEAE Sephacel Chromatography—A column of DEAE Sephacel (5.30 cm²×12 cm)was prepared and equilibrated with Buffer A containing 100 MM NaCl.Fraction III was applied to the column and eluted with a linear gradientfrom 100 mM to 500 MM NaCl. The fractions were analyzed on SDS-PAGE.Fractions containing T7 SSB appeared to be homogeneous as a single bandjudged by electrophoresis under denaturing conditions, but contained alow level of single stranded DNA dependent nucleoside 5′-triphosphataseactivity. The fractions (64 ml) containing the T7 SSB were pooled anddialyzed overnight against Buffer A containing 100 mM NaCl (Fraction V).

Q Sepharose Chromatography—To remove the contaminating ssDNA dependentATPase activity, fraction V was applied to Q Sepharose and eluted with alinear gradient from 100 mM to 500 mM NaCl. The ssDNA dependent ATPaseactivity eluted from the column slightly before the bulk of the SSBprotein. Final fractions of T7 SSB were pooled and dialyzed against 20mM Tris-HCl (pH 7.5); 1 mM EDTA; 0.5 mM DTT; 10 mM NaCl; 50% glyceroland stored at −20° C. (Fraction VI).

Protein Concentration—The protein concentration was determined using theBCA Protein Determination Assay Kit (Pierce, Rockford, Ill.) against aBSA standard curve. After SDS-PAGE electrophoresis of the purified SSBprotein under denaturing conditions, staining with Coomassie Blueproduced a single band corresponding to a molecular weight ofapproximately 30,000. Although the molecular weight of the wild-type T7SSB deduced from the DNA sequence of its gene is 25,562, it migrates asa single band between 25,000 and 31,000 on SDS-PAGE (this aberrantmigration was also observed in Scherzinger et al., 1973, Molecular andGeneral Genetics 123(3):247-262; Reuben and Gefter, 1973, Proceedings ofthe National Academy of Sciences 70(6):1846-1850).

The inventors herein have discovered, surprisingly and unexpectedly,that T7 SSB, both wild-type and mutant varieties, prevent or inhibitprimer extension reactions at lower temperatures (e.g., less than about50° C., and particularly less than about 30° C.) but that suchinhibitive effect is lost at higher, more stringent temperatures (e.g.,greater than about 50° C.). Also, the inventors have discovered,surprisingly and unexpectedly, that inclusion of T7 SSB and/or itsmutant forms in PCR prior to the Initial Denaturation step leads to lessamplification artifacts. This unexpected result is believed to occurbecause nonspecific priming events and/or primer extension products arenot formed or are inhibited from being formed at the lower temperaturesat which PCR mixtures typically are assembled or prepared. Thus, theprimers are sequestered at temperatures where specificity tends to below before the reaction mixture is heated, and then the primers arereleased and thus available for hybridization and polymerization athigher, more stringent temperatures. In this manner, it has beenobserved that amplification of unintended targets formed due to lowhybridization specificity (at low temperature) has been substantiallyreduced.

The following Examples illustrate the effectiveness of a variety of T7SSBs in preventing or inhibiting the generation of nonspecific primerextension products, and are presented by way of illustration and notlimitation.

EXAMPLE 1

A 306 base pair (bp) region of the gene product Numb (sequence providedat SEQ ID NO. 8) was amplified, separately, under a variety of differentconditions of SSB species and concentration, selection of polymerase,etc., as further described below, from 5 nanograms (ng) of human genomicDNA. The target is identified as NT_(—)026437.11 at NCBI (sequencelocation: 54742877 to 54743182). The following amplification primerswere used, each of which was 25 bases in length; Numb Forward:5′-GAGGTTCCTACAGGCACCTGCCCAG-3′ (SEQ ID NO.9) and Numb Reverse:5′-CAAAATCACCCCTCACAGTACTCTG-3′. (SEQ ID NO. 10)

Primers were from standard commercial suppliers and resuspended in TE(10 mM Tris-HCl (pH 8), 1 mM EDTA) at desired concentrations. Humangenomic DNA was from Promega Corporation, Madison, Wis. These primerswere chosen because they have several bases of complementary sequence atthe 3′-end between the forward and reverse primers and generatenonspecific amplification products.

A total of 15 polymerase chain reaction mixtures were assembled at roomtemperature (i.e., 20-25° C.) in 0.5 milliliter (ml) microfuge tubeswith the following general components listed in Table 2 in a finalvolume of 25 microliters (μl): TABLE 2 Volume for 25 μl Final Componentsreaction Concentration Water 19.875 μl   NA 10 × PCR Buffer 2.5 μl 1×  5mM dNTP Mixture 1.0 μl 0.2 mM each dNTP 10 μM Forward and 0.5 μl 0.2 μMor 5 pmol Reverse Primers each/reaction Template DNA 0.5 μl 5ng/reaction SSB, 2 mg/ml 0.5 μl 1 μg/reaction Taq DNA 0.125 μl  0.625units/reaction Polymerase, 5 U/μl

The 15 PCR reaction mixtures had the following specific attributes:

-   -   Reaction 1: antibody-bound Taq DNA Polymerase, no SSB;    -   Reaction 2: chemically-modified Taq DNA Polymerase, no SSB;    -   Reaction 3: unmodified Taq DNA Polymerase, no SSB;    -   Reaction 4: 1 μg wild-type E. coli SSB, antibody-bound Taq DNA        Polymerase    -   Reaction 5: 1 μg wild-type E. coli SSB, chemically-modified Taq        DNA Polymerase;    -   Reaction 6: 1 μg wild-type E. coli SSB, unmodified Taq DNA        Polymerase;    -   Reaction 7: 1 μg wild-type T7 SSB, antibody-bound Taq DNA        Polymerase;    -   Reaction 8: 1 μg wild-type T7 SSB, chemically-modified Taq DNA        Polymerase;    -   Reaction 9: 1 μg wild-type T7 SSB, unmodified Taq DNA        Polymerase;    -   Reaction 10: 1 μg Δ21C T7 SSB, antibody-bound Taq DNA        Polymerase;    -   Reaction 11: 1 μg Δ21C T7 SSB, chemically-modified Taq DNA        Polymerase;    -   Reaction 12: 1 μg Δ21C T7 SSB, unmodified Taq DNA Polymerase;    -   Reaction 13: 1 μg F232L T7 SSB, antibody-bound Taq DNA        Polymerase;    -   Reaction 14: 1 μg F232L T7 SSB, chemically-modified Taq DNA        Polymerase;    -   Reaction 15: 1 μg F232L T7 SSB, unmodified Taq DNA Polymerase.

To minimize pipetting errors, two separate master mixes were assembled.Master Mix 1 was a 6× mix that contained water, PCR buffer, dNTPs, andthe respective polymerase. Master Mix 2 was a 20× mix that contained thehuman genomic DNA and primers. The components were added in thefollowing order to the reaction tubes at room temperature; 23.5 μl ofthe appropriate Master Mix 1 (i.e., with respective polymerase), 0.5 μlof the SSB or SSB Storage Buffer when performing controls, and 1 μl ofMaster Mix 2. It is noted that the concentration of T7 gp2.5 Δ21C usedin Reactions 10-12 was 0.5 mg/ml, not 2 mg/ml as in the other reactionmixtures, and thus 2 μl of this protein were added per 25 μl reactioninstead of 0.5 μl to achieve the same total SSB concentration forReactions 10-12.

The 10×PCR buffer consisted of 10 MM Tris-HCl (pH 8.6), 500 mM KCl, and15 mM MgCl₂. The 5 mM dNTP mixture contained the fourdeoxyribonucleotides required for DNA synthesis (dATP, dGTP, dTTP, anddCTP). The SSBs from T7 were prepared as described elsewhere herein. E.coli SSB and unmodified Taq DNA Polymerase (i.e., non-hot-start) werefrom USB Corporation, Cleveland, Ohio. SSBs were added to the respectivereaction mixtures before the primers and template. For control reactionswithout SSBs, the SSB storage buffer, without SSBs, was added instead.For comparison, two commercially available hot-start products were usedin place of standard (unmodified) Taq DNA Polymerase, Reactions 1, 4, 7,10, and 13 and 2, 5, 8, 11, and 14, respectively. The antibody-bound TaqDNA Polymerase (tradename Platinum™ Taq DNA Polymerase) used inReactions 1, 4, 7, 10, and 13 was from Invitrogen Corporation, Carlsbad,Calif. The chemically-modified Taq DNA Polymerase (tradename HotStarTaq™DNA Polymerase) used in Reactions 2, 5, 8, 11, and 14 was from QiagenIncorporated, Valencia, Calif.

After all the reaction mixtures were completely assembled, they wereincubated at room temperature (i.e., 20-25° C.) for a period of 30minutes before the reactions tubes were placed in the thermal cycler.This extra time at room temperature was chosen so as to favor thegeneration of nonspecific products. Following this room temperatureincubation, reactions tubes were placed in a thermal cycler (MJResearch, Waltham, Mass.) with the following cycling conditions shown inTable 3 common among all the reactions except as otherwise noted: TABLE3 Step Temperature Time Initial 95° C.  2 minutes or 15 Denaturationminutes Denaturation 95° C. 10 seconds Hybridization 63° C. 30 secondsExtension 72° C. 30 seconds Repeat previous three steps 35 times Final72° C.  5 minutes Extension Final Soak 10° C. as necessary

It is noted that the Initial Denaturation time was 2 minutes forreactions containing the unmodified Taq DNA polymerase and theantibody-bound Taq DNA polymerase, and 15 minutes for thechemically-modified Taq DNA polymerase as per the manufacturer'sinstructions.

Following cycling, 10 μl from each of the polymerase chain reactionswere electrophoresed on a 2% TAE agarose gel containing ethidium bromiderun at 100-120 volts for about 1-2 hours in 1×TAE buffer. The primerextension reaction products were visualized using a fluorescent scanner(Hitachi FMBIO II, San Francisco, Calif.).

The results of the foregoing reactions are shown in FIG. 1, wherein thenumbered lanes correspond to the like-numbered Reactions described aboveand the Marker Lane, M, was provided using 1 Kb Plus DNA Ladder fromInvitrogen Corporation, Carlsbad, Calif.

As seen in FIG. 1, the presence of wild-type T7 SSB, as well as the T7SSB mutants referred to herein as Δ21C and F232L markedly improved theyield of specific primer extension products compared to standard Taq DNAPolymerase which does not have a hot-start feature (compares lane 3 tolanes 9, 12, and 15). In the control reaction without SSB (lane 3),primer-dimers are primarily generated at the expense of the specificproduct of 306 bp. Thus, the SSBs reduced or eliminated thesenonspecific primer-dimers and allowed the generation of the specificproduct. In addition, this enhancement effect was shown to becomparable, if not equal, to the two commercially available hot-startpolymerases used in this experiment (compare lanes 1, 2 to lanes 9, 12,and 15). There appeared to be no general effect of adding SSBs intoreactions using polymerases which already included a built-in hot-startfeature (compare lane 1 to lanes 7, 10, and 13 as well as lane 2 tolanes 8, 11, and 14). It is noted that wild-type E. coli SSB (lanes 4-6)completely inhibited the formation of any primer extension products.Thus, wild-type E. coli SSB is unsuitable for use in the present methodsas it inhibits the generation of amplified extension products throughPCR. This experiment demonstrated the effectiveness of not onlywild-type T7 SSB, but also mutant varieties of T7 SSB in which specificamino acids have been changed or deleted.

EXAMPLE 2

This example illustrates the effectiveness of a mixture of wild-type andmutant T7 SSB in the hot-start method. Specifically, this example uses a1:1 mass ratio of wild-type T7 SSB to Δ26C protein in a polymerase chainreaction to reduce the generation of nonspecific primer extensionproducts. In this experiment, 1 microgram (μg) of the mixture contained0.5 μg of each protein. An 1142 base pair (bp) region of the geneproduct p53 (SEQ ID NO. 11) was amplified from either 1 nanogram (ng) or100 picograms (pg) of human genomic DNA. This target is identified asNT_(—)010718.15 at NCBI (sequence location: 7174821 to 7175962). Thefollowing amplification primers were used; p53 Forward:5′-TGCTTTATCTGTTCACTTGTGCCC-3′ (SEQ ID NO. 12) 24 bases in length, andp53 Reverse: 5′-TGTGCAGGGTGGCAAGTGGC-3′ (SEQ ID NO. 13) 20 bases inlength.

Primers were from standard commercial suppliers and resuspended in TE(10 mM Tris-HCl (pH 8), 1 mM EDTA) at desired concentrations. Humangenomic DNA was from Promega Corporation, Madison, Wis. These primerswere chosen because they have several bases of complementary sequence atthe 3′-end between the forward and reverse primers and generatenonspecific amplification products.

A total of 8 polymerase chain reaction mixtures were assembled at roomtemperature (i.e., 20-25° C.) in 0.5 milliliter (ml) microfuge tubeswith the following general components as shown in Table 4 in a finalvolume of 25 microliters (μl): TABLE 4 Volume for 25 μl Final Componentsreaction Concentration Water 20.175 μl   NA 10 × PCR Buffer 2.5 μl 1× 25mM dNTP 0.2 μl 0.2 mM each dNTP Mixture 10 μM Forward and 0.5 μl 0.2 μMor 5 pmol Reverse Primers each/reaction Template DNA 1.0 μl variable SSB0.5 μl variable Taq DNA 0.125 μl  0.625 units/reaction Polymerase, 5U/μl

The 8 PCR reaction mixtures had the following specific attributes:

-   -   Reaction 1: 100 pg genomic DNA, no SSB;    -   Reaction 2: 0.5 μg T7 SSB mix, 100 pg genomic DNA;    -   Reaction 3: 1.0 μg T7 SSB mix, 100 pg genomic DNA;    -   Reaction 4: 2.0 μg T7 SSB mix, 100 pg genomic DNA;    -   Reaction 5: 1 ng genomic DNA, no SSB;    -   Reaction 6: 0.5 μg T7 SSB mix, 1 ng genomic DNA;    -   Reaction 7: 1.0 μg T7 SSB mix, 1 ng genomic DNA;    -   Reaction 8: 2.0 μg T7 SSB mix, 1 ng genomic DNA.

To minimize pipetting errors, three separate master mixes wereassembled. Master Mix 1 was a 10× mix that contained water, PCR buffer,dNTPs, and Taq DNA Polymerase. Master Mix 2 was a 10× mix that containedwater, 100 pg/reaction human genomic DNA, and primers. Master Mix 3 wasa 10× mix that contained water, 1 ng/reaction human genomic DNA, andprimers. It is noted that the final water volume from Table 4 wasdivided such that 48% of the final volume was present in mix 1 and 52%of the final volume was present in mix 2 or mix 3. The components wereadded in the following order to the reaction tubes at room temperature;12.5 μl of Master Mix 1, 0.5 μl of the SSB, or SSB Storage Buffer whenperforming controls, and 12 μl of Master Mix 2 or Master Mix 3 asappropriate.

The 10×PCR buffer consisted of 100 MM Tris-HCl (pH 8.6), 500 mM KCl, and15 mM MgCl₂. The 25 mM dNTP mixture contained the fourdeoxyribonucleotides that are required for DNA synthesis (DATP, dGTP,dTTP, and dCTP). The SSBs from T7 were prepared as described elsewhereherein except the final storage buffer was changed to 20 mM Tris-HCl (pH8.5), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5% Tween-20, and 50%glycerol. Serial dilutions of the SSB mixture were performed in finalstorage buffer in order to add 0.5 μl per reaction. For controlreactions without SSB, the SSB storage buffer, without any SSBs, wasadded instead. SSB was added to the reaction mixture before the primersand template. Serial dilutions of the human genomic DNA were performedin nuclease-free water. Taq DNA Polymerase was from USB Corporation,Cleveland, Ohio.

After the reaction mixtures were completely assembled, the reactiontubes were placed in a thermal cycler (MJ Research, Waltham, Mass.)using the cycling conditions as listed below in Table 5. It is notedthat an additional pre-incubation step at 25° C. for one hour wasprogrammed into the thermal cycler so as to simulate room temperature.This extra time at 25° C. was chosen so as to favor the generation ofnonspecific products. TABLE 5 Step Temperature Time Initial Soak 25° C.60 minutes Initial 95° C.  2 minutes Denaturation Denaturation 95° C. 10seconds Hybridization 60° C.  5 seconds Extension 72° C.  2 minutesRepeat previous three steps 35 times Final 72° C.  5 minutes ExtensionFinal Soak 10° C. as necessary

Following cycling, 10 μl from each of the polymerase chain reactionswere electrophoresed on a 1.5% TAE agarose gel containing ethidiumbromide run at 100-120 volts for about 1-2 hours in 1×TAE buffer. Theprimer extension reaction products were visualized using a fluorescentscanner (Hitachi FMBIO II, San Francisco, Calif.).

The results of the foregoing reactions are shown in FIG. 2, wherein thenumbered lanes correspond to the like-numbered Reactions describedabove, and the Marker Lanes, M, were provided using 1 Kb Plus DNA Ladderfrom Invitrogen Corporation, Carlsbad, Calif.

As seen in FIG. 2, the presence of 1:1 mass ratio mixture of wild-typeT7 SSB to its mutant Δ26C markedly improved the yield of specific primerextension products compared to the control lanes in which no SSB wasintroduced (compare lane 1 to lanes 2, 3, and 4 as well as lane 5 tolanes 6, 7, and 8). The specific product was an 1142 bp fragment of thep53 gene and is indicated by the upper arrow in FIG. 2. At the lowerconcentration of DNA (100 pg), the control reaction (lane 1) did notproduce appreciable specific product but instead primarily producednonspecific product characterized as primer-dimers. At 1 ng of humangenomic DNA, the control reaction did produce some specific product, butalso produced some primer-dimers. The reactions in which the SSB mixturewas present all produced more specific product and reduced or eliminatednonspecific products. One can observe that there is a concentrationdependent effect in which increasing concentrations of SSB (from 0.5 μgto 2 μg) yielded increasing amounts of specific product (e.g., comparelanes 2, 3, and 4). This was believed due to the stoichiometry of SSBbinding to the primers in the reaction. This effect will be elaboratedon in a later example.

EXAMPLE 3

This example further illustrates the effectiveness of mixtures ofwild-type and mutant T7 SSB in blocking primer extension at roomtemperature. Specifically, this example uses a 1:1 mass ratio ofwild-type T7 SSB to Δ26C protein in a ‘mock’ polymerase chain reactionin which primer extension was directed at two primers that werepurposefully designed to form hybrids. Thus, there was no exogenousdsDNA template in the reaction, only the primers themselves serve astemplate for synthesis. This assay was designed to access the ability ofSSB to block DNA synthesis from an extendable hybrid at twotemperatures. The first temperature was room temperature (25° C.), asthis simulated the temperature at which reactions are generallyassembled. The second was at 72° C., which is a more optimal temperaturefor DNA synthesis by Taq DNA Polymerase.

The two primers chosen for this experiment were designed to form a dsDNAhybrid with 14 bp of overlap at their 3′-ends. The forward primer of 23bases had a HEX fluorescent label attached to its 5′end which enableddetection of the synthesis product on a fluorescent scanner. Since thereverse primer of 41 bases has 14 bp of overlap with the forward primer,the maximum synthesis product that could be generated from the forwardprimer was 50 bases. This primer extension product was visualized duringdenaturing polyacrylamide gel electrophoresis. A schematic of the assayis shown in FIG. 3 a.

In this assay, 1 pmol of each primer was placed in a 10 μl reactionvolume and tested against several concentrations of the SSB mixture. Theprimer sequences were as follows; Forward:5′-[HEX]-CTTTTCCCAGTCACGACGTTGTA-3′ (SEQ ID NO. 14) 23 bases in length.and Reverse: 5′-ATGCAAGCTTGGCACTGGCCGTCGTTTTACA (SEQ ID NO. 15)ACGTCGTGAC-3′ 41 bases in length.

Primers were from standard commercial suppliers and resuspended in TE(10 mM Tris-HCl (pH 8), 1 mM EDTA) at desired concentrations. A total of10 mock polymerase chain reaction mixtures were assembled at roomtemperature (i.e., 20-25° C.) in 0.5 milliliter (ml) microfuge tubeswith the following general components as listed in Table 6 in a finalvolume of 10 microliters (μl): TABLE 6 Volume for 10 μl Final Componentsreaction Concentration Water 8.27 μl NA 10 × PCR Buffer 1.0 μl 1× 25 mMdNTP 0.08 μl 0.2 mM each dNTP Mixture 10 μM Forward and  0.1 μl 0.1 μMor 1 pmol Reverse Primers each/reaction SSB  0.5 μl variable Taq DNA0.05 μl 0.25 units/reaction Polymerase, 5 U/μl or none

The 10 mock PCR reaction mixtures had the following specific attributes:

-   -   Reaction 1: no SSB, no Taq DNA Polymerase, 25° C. incubation;    -   Reaction 2: Taq DNA Polymerase, no SSB, 25° C. incubation;    -   Reaction 3: 0.5 μg T7 SSB mix, Taq DNA Polymerase, 25° C.        incubation;    -   Reaction 4: 1.0 μg T7 SSB mix, Taq DNA Polymerase, 25° C.        incubation;    -   Reaction 5: 2.0 μg T7 SSB mix, Taq DNA Polymerase, 25° C.        incubation;    -   Reaction 6: no SSB, no Taq DNA Polymerase, 72° C. incubation;    -   Reaction 7: Taq DNA Polymerase, no SSB, 72° C. incubation;    -   Reaction 8: 0.5 μg T7 SSB mix, Taq DNA Polymerase, 72° C.        incubation;    -   Reaction 9: 1.0 μg T7 SSB mix, Taq DNA Polymerase, 72° C.        incubation;    -   Reaction 10: 2.0 μg T7 SSB mix, Taq DNA Polymerase, 72° C.        incubation.

To minimize pipetting errors, three separate master mixes wereassembled. Master Mix 1 was a 1 2X mix that contained water, PCR buffer,dNTPs, and Taq DNA Polymerase. Master Mix 2 was a 12× mix that containedwater, PCR buffer, dNTPs, but no Taq DNA Polymerase. Master Mix 3 was a12× mix that contained water and primers. It is noted that the finalwater volume from Table 6 was divided such that 46.8% of the finalvolume was present in mix 1 or mix 2 and 53.2% of the final volume waspresent in mix 3. The components were added in the following order tothe reaction tubes at room temperature; 5.0 μl of Master Mix 1 or MasterMix 2 as appropriate, 0.5 μl of the SSB or SSB Storage Buffer whenperforming controls, and 4.5 μl of Master Mix 3.

The 10×PCR buffer consisted of 10 mM Tris-HCl (pH 8.6), 500 mM KCl, and15 mM MgCl₂. The 25 mM dNTP mixture contained the fourdeoxyribonucleotides that are required for DNA synthesis (dATP, dGTP,dTTP, and dCTP). The SSBs from T7 were prepared as described elsewhereherein except the final storage buffer was changed to 20 mM Tris-HCl (pH8.5), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5% Tween-20, and 50%glycerol. Serial dilutions of the SSB mixtures were performed in finalstorage buffer in order to add 0.5 μl per reaction. For controlreactions without SSB, the SSB storage buffer, without any SSBs, wasadded instead. SSB was added to the reaction mixture before the primers.Negative control reactions without Taq DNA Polymerase (i.e., those thatwould be a baseline to judge primer extension product yields) had thebalance made up with water. Taq DNA Polymerase was from USB Corporation,Cleveland, Ohio.

After the reaction mixtures were completely assembled, the reactiontubes were placed in a thermal cycler (MJ Research, Waltham, Mass.). Oneset of identical reactions was subjected to 25° C. for four hours toover-estimate the amount of time required to assemble PCR reactions. Theother identical set was subjected to 15 cycles at 25° C. for 15 secondsand 72° C. for 15 seconds to provide ideal synthesis conditions for TaqDNA Polymerase and to determine if the SSBs were still inhibitory.Following these incubations, the reactions were stored at 4° C. or onice until required. In order to visualize primer extension products, 0.5μl (0.05 pmol of each primer) of each reaction were electrophoresed on a15% (29:1) denaturing polyacrylamide gel with 42% urea. The gel was castwith 1 mm spacers in 1×GTG buffer (USB Corporation, Cleveland, Ohio) andrun at a constant power of 6 watts per gel until a tracer dye(Bromo-cresol Green) had run about 75% the length of the gel (about 25minutes). The primer extension reaction products were visualized using afluorescent scanner (Hitachi FMBIO II, San Francisco, Calif.).

The results of the foregoing reactions are shown in FIG. 3 b, whereinthe numbered lanes correspond to the like-numbered Reactions describedabove.

As seen in FIG. 3 b, following four hours of incubation at 25° C., TaqDNA Polymerase yields a primer extension product of 50 bases from theprimer-hybrid compared to the negative control in which no polymerasewas present in the reaction (compare lane 1 to lane 2). In addition, atthe 3 concentrations of SSB tested, the presence of the 1:1 mass ratiomixture of wild-type T7 SSB to its mutant Δ26C blocked synthesis fromthe primer-hybrid at 25° C. comparable to the negative control (comparelanes 1 and 2 to lanes 3-5). In contrast, using a 72° C. incubationtemperature yielded reaction products that were of similar yields in allof the lanes that included Taq DNA Polymerase, even when SSBs wereincorporated into the reaction mixture (compare lanes 6-10). The factthat primer extension could take place at elevated, or stringent,temperatures demonstrated the blocking effect of the single-strandedbinding proteins had been terminated. This experiment confirmed severaldesirable attributes of SSBs for use in the methods described herein: 1)interaction with ssDNA at lower temperatures at which reactions areconventionally assembled effective to inhibit the generation ofextension products at those temperatures; 2) such interaction with ssDNAin conventional PCR buffers; and 3) termination of this interaction withssDNA at more stringent temperatures.

EXAMPLE 4

This example illustrates a useful range of effective concentrations ofT7 SSB that achieve the desired effect of reducing the generation ofnonspecific primer extension products. The following experiment wasdesigned taking into account both a) the stoichiometric binding ratio ofT7 SSB of 7 nucleotides bound per protein monomer, and b) the totalamount of primers (ssDNA) in a given reaction. The experiment was anamplification of the Numb target of 306 bp from 1 ng of human genomicDNA that was used in Example 1. The primers were each 25 bases in lengthas follows: Numb Forward: 5′-GAGGTTCCTACAGGCACCTGCCCAG-3′ (SEQ ID NO. 8)and Numb Reverse: 5′-CAAAATCACCCCTCACAGTACTCTG-3′. (SEQ ID NO. 9)

Primers were from standard commercial suppliers and resuspended in TE(10 mM Tris-HCl (pH 8), 1 mM EDTA) at desired concentrations. Humangenomic DNA was from Promega Corporation, Madison, Wis. Recall, theseprimers were chosen because they have several bases of complementarysequence at the 3′-end between the forward and reverse primers andgenerate nonspecific amplification products.

A total of 7 polymerase chain reaction mixtures were assembled at roomtemperature (i.e., 20-25° C.) in 0.5 milliliter (ml) microfuge tubeswith the following general components as listed in Table 7 in a finalvolume of 25 microliters (μl): TABLE 7 Volume for 25 μl Final Componentsreaction Concentration Water 20.175 μl   NA 10 × PCR Buffer 2.5 μl 1× 25mM dNTP 0.2 μl 0.2 mM each dNTP Mixture 10 μM Forward and 0.5 μl 0.2 μMor 5 pmol Reverse Primers each/reaction Template DNA 1.0 μl 1ng/reaction SSB 0.5 μl variable Taq DNA 0.125 μl  0.625 units/reactionPolymerase, 5 U/μl

The 7 PCR reaction mixtures had the following specific attributes:

-   -   Reaction 1: no SSB;    -   Reaction 2: 0.0625 μg wild-type T7 SSB;    -   Reaction 3: 0.125 μg wild-type T7 SSB;    -   Reaction 4: 0.25 μg wild-type T7 SSB;    -   Reaction 5: 0.5 μg wild-type T7 SSB;    -   Reaction 6: 1.0 μg wild-type T7 SSB;    -   Reaction 7: 2.0 μg wild-type T7 SSB.

To minimize pipetting errors, two separate master mixes were assembled.Master Mix 1 was a 10 × mix that contained water, PCR buffer, dNTPs, andTaq DNA Polymerase. Master Mix 2 was a 10× mix that contained water, 1ng/reaction human genomic DNA, and primers. It is noted that the finalwater volume from Table 7 was divided such that 48% of the final volumewas present in mix 1 and 52% of the final volume was present in mix 2.The components were added in the following order to the reaction tubesat room temperature; 12.5 μl of Master Mix 1, 0.5 μl of the SSB or SSBStorage Buffer when performing controls, and 12 μl of Master Mix 2.

The 10×PCR buffer consisted of 100 mM Tris-HCl (pH 8.6), 500 mM KCl, and15 mM MgCl₂. The 25 mM dNTP mixture contained the fourdeoxyribonucleotides that are required for DNA synthesis (dATP, dGTP,dTTP, and dCTP). Wild-type T7 SSB was prepared as described elsewhereherein except the final storage buffer was changed to 20 mM Tris-HCl (pH8.5), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5% Tween-20, and 50%glycerol. Serial dilutions of the wild-type T7 SSB were performed infinal storage buffer in order to add 0.5 μl per reaction. For controlreactions without SSB, the SSB storage buffer, without any SSBs, wasadded instead. SSB was added to the reaction mixture before the primersand template. Taq DNA Polymerase was from USB Corporation, Cleveland,Ohio.

Each reaction contained 5 picomoles (pmol) of each primer and thereforea relatively simple calculation could be performed to determine themolar amount of single-stranded DNA binding sites in the reaction. Sincethe primers were each 25 bases in length and T7 SSB binds about 7nucleotides per protein monomer, each primer had about 3.57 bindingsites. Given there were 10 pmol total primers in each reaction×3.57binding sites per primer meant there were roughly 36 pmol total ssDNAbinding sites in each reaction. The mass amount of wild-type T7 SSBvaried in this experiment, serially-doubling from 62.5 ng per reactionto 2 μg per reaction. Given the molecular weight of T7 SSB is 25,562 gmper mol per monomer, the following Table 8 could be constructed showingthe molar amount of T7 SSB monomers in each reaction condition as wellas the molar ratio of T7 SSB to total available binding sites in eachreaction condition. TABLE 8 Molar amount of Molar ratio of Mass amountof T7 SSB monomers monomer:ssDNA 62.5 ng  2.44 pmol 0.068 125 ng  4.89pmol 0.137 250 ng  9.78 pmol 0.274 500 ng 19.56 pmol 0.548 1 μg 39.12pmol 1.096 2 μg 78.24 pmol 2.192

From Table 8, it is clear that the lowest concentration of T7 SSB (62.5ng) in any reaction was an order of magnitude less than the molar amountof available binding sites in the reaction. The transition point fromthe lowest concentration of T7 SSB to one in which the molar ratio wasequivalent occurred around 1 μg of T7 SSB. Thus, for primers 25 bases inlength and at 5 pmol each in the reaction, concentrations of T7 SSB thatwere greater than 1 μg per reaction were in molar excess over theavailable ssDNA binding sites; as will be seen these were preferredconditions. It is noted that the molar ratios of T7 SSB (or any SSB witha known molecular weight) to available binding sites can be determinedfor a variety of primers of differing lengths and concentrations throughthese relatively straightforward calculations.

After all the reaction mixtures were completely assembled, the reactiontubes were placed in a thermal cycler (MJ Research, Waltham, Mass.)using the cycling conditions as listed below in Table 9. It is notedthat an additional pre-incubation step at 25° C. for one hour wasprogrammed into the thermal cycler so as to simulate room temperature.This extra time at 25° C. was chosen so as to favor the generation ofnonspecific products. TABLE 9 Step Temperature Time Initial Soak 25° C.60 minutes Initial 95° C.  2 minutes Denaturation Denaturation 95° C. 10seconds Hybridization 60° C.  5 seconds Extension 72° C. 30 secondsRepeat previous three steps 35 times Final 72° C.  5 minutes ExtensionFinal Soak 10° C. as necessary

Following cycling, 10 μl from each of the polymerase chain reactionswere electrophoresed on a 1.5% TAE agarose gel containing ethidiumbromide run at 100-120 volts for about 1-2 hours in 1×TAE buffer. Theprimer extension reaction products were visualized using a fluorescentscanner (Hitachi FMBIO II, San Francisco, Calif.).

The results of the foregoing reactions are shown in FIG. 4, wherein thenumbered lanes correspond to the like-numbered Reactions described aboveand the Marker Lane, M, was provided using 1 Kb Plus DNA Ladder fromInvitrogen Corporation, Carlsbad, Calif.

As is shown in FIG. 4, wild-type T7 SSB markedly enhanced the yield ofthe specific product. There is a clear concentration effect in whichincreasing concentrations of SSB yielded not only more specific productbut fewer primer-dimers. This was exemplified by the reaction shown inlane 7 which had the lowest amount of primer-dimers and the highestamount of specific product relative to the control reaction without SSB(lane 1). This concentration effect was consistent with thestoichiometry predictions previously described. In reactions in whichmolar ratios of SSB monomers to available ssDNA binding sites wassignificantly less than one (lanes 2-4) less specific product wasgenerated. In reactions in which molar ratios of SSB monomers toavailable ssDNA binding sites was close to or greater than one (lanes5-7), more specific product was generated. Thus, although a range ofconcentrations of SSB are effective at increasing specific productyield, those concentrations that are equal to or exceed the molarconcentration of primers in the reaction are most preferred.

One advantage of the primer sequestration method described herein isthat it will work with any polymerase because the SSB interacts with andacts to inhibit the primers, and does not depend on any interaction witha particular polymerase. The antibody and chemical methods discussed inthe BACKGROUND section require modifications to individual polymerases.There are at least 10 different polymerases commonly used for PCR, andthus the present invention has much broader utility. Furthermore, likethose other methods, the methods disclosed herein permit the completereaction system, including all of the reagents necessary to carry outmultiple cycles of hybridization and primer extension reactions, to becompletely assembled at nonstringent temperatures (such as roomtemperature), without the need to subsequently add a polymerase or anyother component to the reaction mixture, thus risking contamination ofthe reactions.

As noted above, while the foregoing description has been provided in thecontext of performing a polymerase chain reaction, the invention is notto be limited to PCR. SSBs can be incorporated into other reactionmixtures for duplicating a template nucleic acid via primer-templatehybridization and extension reactions to inhibit or prevent nonspecificprimer extension products where it is convenient to combine all thecomponents necessary for both reactions at nonstringent temperatures.

Also provided is a storage buffer solution useful for long-term storageof the SSBs useful in the disclosed methods (preferably up to one year)so that they do not lose their functional activity; i.e., their abilityto effectively sequester primers or prevent or inhibit the generation ofnonspecific primer extension products according to methods describedherein. The storage buffer solution preferably has the followingcomponents listed in Table 10. It is noted that in Table 10, anyconcentration or range for any one component can be combined with anyconcentration or range for any other component to provide the buffersolution; it is not necessary that all concentrations or ranges comefrom the same column. TABLE 10 Buffer solution for storage of SSBComponent Preferred Less Preferred Tris HCl, pH 7.5 20 mM 1-100 mM 5-80mM 10-60 mM 15-40 mM EDTA 1 mM 1-100 mM 1-50 mM 1-10 mM DTT 0.5 mM0.005-200 mM 0.01-100 mM 0.02-50 mM 0.03-25 mM 0.04-10 mM Salt (pref.NaCl) 10 mM 5-80 mM 8-60 mM 10-50 mM 10-20 mM 0 mM (for particularembodiment, explained below) Glycerol 50 mass percent 10 to 80 masspercent 20 to 70 mass percent 30 to 60 mass percent Water BalanceBalance

A suitable storage buffer can be prepared for, e.g., wild-type T7 gp2.5using no salt, i.e., no sodium chloride. This was a particularlysurprising and unexpected result, as it ordinarily would have beenexpected that to prevent the SSB from precipitating out of solution, aquantity of salt, such as sodium chloride, would be required. Generally,it is preferred nevertheless to provide the buffer solution with 10 mMsalt concentration. For the T7 gp2.5-Δ21 C mutant disclosed above, asomewhat higher salt concentration is desirable to sustain the mutant insolution (i.e., prevent its precipitation), and preferably greater than50 mM salt concentration is used.

To perform a PCR amplification procedure using the SSB in its storagebuffer, an aliquot of the SSB in its storage buffer is extracted, as bypipette, from the buffer solution container and then delivered to thePCR reaction vessel or tube when preparing the PCR reaction mixture,typically at room temperature. It has been found that the buffersolution disclosed above does not adversely affect the PCR amplificationmechanism, and that suitable amplification results are obtained using avariety of different polymerases, e.g., with wild-type Taq DNApolymerase and a mutant variant of Taq DNA polymerase as well as Pfu DNApolymerase.

Thus, the buffer solution disclosed herein has the advantages the SSBsremain stable and functionally active (capable to inhibit primerhybridization at non-stringent temperatures) when stored therein forextended periods, preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11or 12, months, and the residual storage buffer solution that isdelivered to the PCR reaction tube along with the SSBs does notadversely affect PCR amplification reactions when using a range ofpolymerases.

Preferably, a liquid formulation composed of an SSB, e.g., wild-typeT7gp2.5, in a buffer solution as described above has a total proteinconcentration ranging from 1 μg/ml to 200 mg/ml, more preferably 10μg/ml to 100 mg/ml, even more preferably 100 μg/ml to 50 mg/ml and mostpreferably between 1 mg and 5 mg/ml. In addition, other single-strandednucleic acid binding proteins involved or not involved in replicationmechanism such as but not limited to the following SSBs can be used incombination with or in place of wild-type T7gp2.5: T7gp2.5-F232L, T7gp2.5-Δ21C, T4 gp32, Rec A, λ beta protein, etc. In a preferredembodiment, the resulting formulation of wild-type T7 gp2.5 (or other)binding protein in the above-described storage buffer has a pH between4.0 and 12.0, more preferably between pH 6.0 and 10.0, even morepreferably between 7.0 and 9.0 and most preferably pH 7.5±0.2. Table 11below describes preferred compositions for a formulation of SSB in astorage buffer which can include further or additional additives orcomponents beyond those described above. TABLE 11 Compositions for SSBformulations in storage buffer Most preferred Component/Propertyconcentration/value More Preferably Preferably Less Preferably SSB(preferably T7gp2.5, 1-5 mg/ml 100 μg-50 mg/ml 10 μg-100 mg/ml 1 μg-200mg/ml wild-type or mutant variant) pH 7.5 ± 0.2 7.0-9.0 6.0-10.04.0-12.0 Buffer such as MOPS, 20 mM ± 5 mM Tris-HCl pH 15-50 mM 5-100 mM0-250 mM HEPES, TRICINE, etc 7.5 to pH 8.5 Reducing Agent (DTT or β- 1 ±0.2 mM 0.5-10 mM 0.1-50 mM 0-100 mM ME) Monovalent Ions (Na⁺, K⁺, 10 ± 2mM 1-100 mM 0.5-200 mM 0-500 mM Li⁺, Cl⁻, etc.) Complexing/ChelatingAgent 0.5 mM ± 0.1 mM 0.1-1 mM 0.01-2 mM 0-100 mM such as EDTA, EGTA,etc. Divalent Ions (Zn²⁺, Mg²⁺, 0-50 mM 0-100 mM 0-200 mM 0-500 mM Co²⁺,etc.) Amino Acid Based Carrier 0-1 mg/ml 0-10 mg/ml 0-100 mg/ml such asBovine Serum Albumine, Poly L-lysine, etc. Non ionic Detergents such as0.1%-1% (v/v) 0.01%-5% (v/v) 0-20% (v/v) Nonidet P40, Triton X100, Tween20, etc. Zwitterionic Detergents such 0.1%-1% (v/v) 0.01%-5% (v/v) 0-20%(v/v) as CHAPS or CHAPSO Ionic Detergents such as SDS 0.005%-0.1% (v/v)0.0001%-1% (v/v) 0-5% (v/v) DMSO 0.01%-1% (v/v) 0.001%-10% (v/v) 0-50%(v/v) Polysaccharide/Dextran 1%-5% (v/v) 0.1%-10% (v/v) 0-50% (v/v)Protein Stabilizer such as 50% ± 5% (v/v) 5%-65% (v/v) 1%-75% (v/v)0-99% (v/v) glycerol, Ethylene glycol, etc. Mono or disaccharide such as10-10,000 × Protein 1-100 × Protein mass 0.1-10 × protein glucose,maltose, etc. mass mass

Although the hereinabove described embodiments constitute preferredembodiments of the invention, it is to be understood that variousmodifications or changes can be made thereto without departing from thespirit and the scope of the invention as set forth in the appendedclaims.

1. A method of duplicating a template nucleic acid, or a portionthereof, wherein a primer having a nucleotide sequence that iscomplementary to a target portion of the template nucleic acid ishybridized to the template nucleic acid and then extended via an enzyme,the method comprising the steps of: (a) at a first temperature,preparing a reaction mixture comprising a primer, a template nucleicacid, an enzyme effective to catalyze primer extension and an effectiveamount of single-stranded nucleic acid binding protein, (b) at a secondtemperature higher than said first temperature, carrying out ahybridization reaction to produce a hybridized product, and (c) at athird temperature higher than said first temperature, carrying out aprimer extension reaction to produce from said hybridized product anextended product; wherein the generation of specific extended product isimproved as a result of incorporating said single-stranded nucleic acidbinding protein into said reaction mixture at said first temperature. 2.A method according to claim 1, said enzyme being a polymerase, saidreaction mixture further comprising a divalent cation at said firsttemperature.
 3. A method according to claim 1, wherein saidsingle-stranded nucleic acid binding protein inhibits the generation ofnonspecific primer extension products at said first temperature.
 4. AMethod according to claim 3, wherein at said second temperature saidprimer is substantially uninhibited by said single-stranded nucleic acidbinding protein from participating in the hybridization reaction.
 5. Amethod according to claim 4, wherein said primer becomes uninhibitedfrom participating in the hybridization reaction as a result of or inconjunction with said single-stranded nucleic acid binding protein beingdenatured at said second temperature.
 6. A method according to claim 3,wherein at said third temperature said primer is uninhibited by saidsingle-stranded nucleic acid binding protein from participating in theprimer extension reaction.
 7. A method according to claim 1, said firsttemperature being at or below 37° C. and said second and thirdtemperatures each being in the range of 50° C. to about 72° C.
 8. Amethod according to claim 1, further comprising the following stepperformed intermediate said steps (a) and (b): (a.1) initially heatingthe reaction mixture to a fourth temperature, higher than said secondand third temperatures, to denature double-stranded template nucleicacids present in the reaction mixture.
 9. A method according to claim 1,further comprising the following step performed subsequent to said step(c): (d) heating the reaction mixture to a fourth temperature, higherthan said second and third temperatures, to denature double-strandedextended products present in the reaction mixture which were producedduring said step (c).
 10. A method according to claim 9, comprisingcarrying out an amplification reaction by repeating said steps (b) and(c) at least once to generate an amplified product, wherein thegeneration of specific amplified product is improved as a result ofincorporating said single-stranded nucleic acid binding protein intosaid reaction mixture at said first temperature.
 11. A method accordingto claim 10, said first temperature being at or below 37° C., saidsecond and third temperatures each being in the range of 50° C. to about72° C., and said fourth temperature being at or above 90° C.
 12. Amethod according to claim 10, said second and third temperatures beingthe same.
 13. A method according to claim 1, wherein saidsingle-stranded nucleic acid binding protein does not have any knownenzymatic activity.
 14. A method according to claim 1, saidsingle-stranded nucleic acid binding protein comprising at least one ofwild-type T7 gp2.5 and its mutant variants, or a combination thereof.15. A method according to claim 1, said single-stranded nucleic acidbinding protein comprising T7 gp2.5-F232L.
 16. A method according toclaim 1, said single-stranded nucleic acid binding protein comprising T7gp2.5-Δ21C.
 17. A method according to claim 1, said single-strandednucleic acid binding protein comprising a mixture of proteins includingwild-type T7 gp2.5 and T7 gp2.5-Δ26C.
 18. A method according to claim 1,excluding the introduction of any additional reagent to the reactionmixture subsequent to the reaction mixture being prepared at said firsttemperature.
 19. A method according to claim 1, wherein thestoichiometric ratio of single-stranded nucleic acid binding proteinmolecules to primer molecules in said reaction mixture is greater thanor equal to
 1. 20. A method according to claim 19, wherein saidstoichiometric ratio is greater than or equal to
 2. 21. A methodaccording to claim 1, wherein said single-stranded nucleic acid bindingprotein is supplied from a composition of it in a buffer solution, saidbuffer solution comprising 1-100 mM Tris-HCl pH 7.5, 1-100 mM EDTA,0.005-200 mM DTT, 10-80 mass percent glycerol, balance water.
 22. Aprimer complex comprising: a primer having a nucleotide sequence that iscomplementary to a specific target portion of a template nucleic acidmolecule, and a single-stranded nucleic acid binding protein interactingwith said primer; said single-stranded nucleic acid binding proteinbeing selected such that 1) it in effect inhibits said primer fromparticipating in a primer extension reaction up to at least a firsttemperature at or below 30° C., and 2) said interaction ceases or isdisrupted at a second temperature in the range of 30° C. to about 72° C.such that said primer is substantially uninhibited by saidsingle-stranded nucleic acid binding protein from participating in aprimer extension reaction at said second temperature.
 23. A complexaccording to claim 22, said second temperature being in the range ofabout 50° C. to about 72° C.
 24. A complex according to claim 22, saidsingle-stranded nucleic acid binding protein being bound to said primervia a non-covalent bond.
 25. A complex according to claim 22, saidsingle-stranded nucleic acid binding protein being selected such that itin effect inhibits said primer from participating in a primer extensionreaction up to at least 50° C., wherein its inhibitive capability isdestroyed following incubation at 90° C.
 26. A complex according toclaim 22, wherein the inhibitive capability of said single-strandednucleic acid binding protein is destroyed following incubation at 90° C.27. A complex according to claim 22, said single-stranded nucleic acidbinding protein comprising wild-type T7 gp2.5 or a mutant variant, or acombination thereof.
 28. A complex according to claim 22, saidsingle-stranded nucleic acid binding protein comprising wild-type T7gp2.5.
 29. A complex according to claim 22, said single-stranded nucleicacid binding protein comprising T7 gp2.5-F232L.
 30. A complex accordingto claim 22, said single-stranded nucleic acid binding proteincomprising T7 gp2.5-Δ21C.
 31. A complex according to claim 22, saidsingle-stranded nucleic acid binding protein comprising a mixture ofproteins including wild-type T7 gp2.5 and T7 gp2.5-Δ26C.
 32. A complexaccording to claim 22, said single-stranded nucleic acid binding proteinbeing bound to said primer via a thermolabile bond that is spontaneouslyseverable at said second temperature, in the range of about 50° C. toabout 72° C.
 33. A complex according to claim 22, wherein saidsingle-stranded nucleic acid binding protein is denatured at said secondtemperature such that said interaction is disrupted as a result of or inconjunction with the denaturation of said single-stranded nucleic acidbinding protein.
 34. A PCR reaction mixture comprising a primer having anucleotide sequence that is complementary to a specific target portionof a template nucleic acid, and a single-stranded nucleic acid bindingprotein effective to inhibit said primer from participating in a primerextension reaction up to at least a first temperature at or below 30°C., wherein the inhibitive capability of said single-stranded nucleicacid binding protein is lost at a second temperature in the range of 30°C. to about 72° C.
 35. A PCR reaction mixture according to claim 34,said second temperature being in the range of about 50° C. to about 72°C.
 36. A PCR reaction mixture according to claim 34, saidsingle-stranded nucleic acid binding protein comprising at least one ofwild-type T7 gp2.5 and its mutant variants, or a combination thereof.37. A PCR reaction mixture according to claim 34, said single-strandednucleic acid binding protein comprising at least one of T7gp2.5-F232Land T7 gp2.5-Δ21C.
 38. A PCR reaction mixture according to claim 34,said single-stranded nucleic acid binding protein comprising a mixtureof proteins including wild-type T7 gp2.5 and T7 gp2.5-Δ26C.
 39. A PCRreaction mixture according to claim 34, wherein the stoichiometric ratioof single-stranded nucleic acid binding protein molecules to primermolecules in said PCR reaction mixture is greater than or equal to 1.